Pregnane X receptor agonist nomilin extends lifespan and healthspan in preclinical models through detoxification functions

Citrus fruit has long been considered a healthy food, but its role and detailed mechanism in lifespan extension are not clear. Here, by using the nematode C. elegans, we identified that nomilin, a bitter-taste limoloid that is enriched in citrus, significantly extended the animals’ lifespan, healthspan, and toxin resistance. Further analyses indicate that this ageing inhibiting activity depended on the insulin-like pathway DAF-2/DAF-16 and nuclear hormone receptors NHR-8/DAF-12. Moreover, the human pregnane X receptor (hPXR) was identified as the mammalian counterpart of NHR-8/DAF-12 and X-ray crystallography showed that nomilin directly binds with hPXR. The hPXR mutations that prevented nomilin binding blocked the activity of nomilin both in mammalian cells and in C. elegans. Finally, dietary nomilin supplementation improved healthspan and lifespan in D-galactose- and doxorubicin-induced senescent mice as well as in male senescence accelerated mice prone 8 (SAMP8) mice, and induced a longevity gene signature similar to that of most longevity interventions in the liver of bile-duct-ligation male mice. Taken together, we identified that nomilin may extend lifespan and healthspan in animals via the activation of PXR mediated detoxification functions.


1.
To solidify the effect of NML through IIS and the NHRs, it would be important to check for the expression of known DAF-16 target genes. Does NML affect dauer formation? 2. For the longevity assay statistics, it would be important to show the number of censored worms. Also, while the authors use large numbers of worms in each experiment, it would be important to do independent biological repeats of the lifespan assays. 3. To solidify the survival experiments, it would be useful to use limonin as a negative control. 4. I see a few inconsistencies in the lifespan data in Fig 4. The lifespan extension in the WT is smaller than that reported in Fig 1. While I really like the idea of testing hPXR in the worm, I do not see the evidence in the data that it can replace NHR-8 or DAF-12. To look at this, there should be a comparison between the "rescue" strains with WT and mutant hPXR in Table S6. 5. What is the effect of hPXR expression on the target genes of NHR-8 and/or DAF-12? This would be important to look at to support the notion that hPXR can substitute for the NHRs. 6. In Fig 4H, the various hPXR mutants are analyzed are used for NML and RIF treatments. It would be great if the authors could comment on the fact that all mutants have a lower baseline activity. Would this suggest that the compounds stabilize the protein? 7. Fig 5 shows a strong and consistent effect of NML in vivo. While interestingly, this would need to be done in a PXR mutant to suggest that NML uses the detoxification pathway in the context of neuroprotection.
Minor points 1. While the language if clear, I would suggest for a native speaker to edit the manuscript. 2. I would recommend refraining from using the term "anti-aging". This is not very well defined, particularly in the context of the paper where survival is only measured in the worm. The term better belongs into popular culture. 3. Also from the data presented here, I think one cannot conclude any recommendations or comments regarding citrus consumption.
Reviewer #2 (Remarks to the Author): In manuscript "Pregnane X receptor agonist nomilin extends lifespan and healthspan through the detoxification functions", the authors show through a combination of X-ray structure, in vitro and in vivo (in nematode and mouse) experiments that nomilin, a compound found in citrus fruits, is able to activate in a beneficial way for the organism the nuclear receptors NHR8 and DAF12 in worms, as well as their couterpart in mammals, PXR. PXR is a receptor rather known for its adverse effects following the binding of exogenous molecules, whereas here the authors show that nomilin can increase lifespan and healthspan via PXR. From a technical point of view, the biological experiments are numerous and varied, and are rigorously carried out with all necessary controls. The accomplished work seems dense and of quality, even if the authors sometimes get confused in the text, especially for the notations of the mutants. However the quality of the X-ray structure is a major issue. It can be improved and needs to be re-refined. My comments will essentially focus on the structural aspects of this manuscript, even though I have some general comments, listed below, to improve the readability : 1/ Please specify the origin of PXR in the whole manuscript, by writing mPXR (for mouse) or hPXR (for human) depending on the sentence or figure legend. 2/ Fig. 4 : panels D, E, F, and G are not easy to read, please increase the size of residue labels, and potentially the size of these panels. In G, write the name of the compounds with the same color code as the sticks and place the labels in a more obvious way. In H, there is a mistake, M245Q should be M425Q, and M247A should be S247R. In C, please label the b1' strand as it is cited in the text. Molecules in panel A are too small to be read correctly. 3/ Fig. 6 : panel H, increase the font size. 4/ Please standardize the nomenclature for residues as both « Ser247 and Gln285 » and « M243, W299, I414, and M425 » are found in the manuscript. 5/ Fig. S1 : some panels should be more described, for example add DAY 10 on a and b, DAY 15 on c and d, DAY5 on e and DAY10 on f. 6/ In « Crystal structure of nomilin-PXR complex indicates critical amino acids for the binding affinity » section, in last sentence, M247A/R should be S247A/R. 7/ In « Mammalian PXR is a functional ortholog of NHR-8 /DAF-12 » section there is a mistake on mutant that should be hPXR S247R (I expect) 8/ Maybe I missed something, but in « Dissussion », what are AD, PD and CR ?
Here are my scientific comments : 9/ For TR-FRET assay, why the authors used the T0901317 LXR agonist as a reference instead of rifampicine or SR12813 that are commonly used as PXR reference ligands ? Is this compound known to be a specific PXR agonist ? In Fig. 4 B, the concentration should be stated as a power of 10 for easier reading. Could the authors mention and discuss the affinity values (Ki values) of the different compounds in the text ? Finally, in Fig. S3, could the authors add the equivalent panel to those in a-g for PXR ?
10/ The conclusion of the « Mammalian PXR is a functional ortholog of NHR-8 /DAF-12 » section -« These data indicate that hPXR could partially compensate the function of NHR-8 and DAF-12 in mediating nomilin dependent lifespan-extending effects in C. elegans » -implies that hPXR could recognize the response elements of NHR8 and DAF-12. Out of curiosity, are these DNA sequences known in nematode ? 11/ Concerning the structure, it was not clear for me if the authors were able to model the ligand in both chains of PXR structure before reading the PDB validation report and having a look to the structure.
Please be more precise in text. However, several points let me think that this structure should be improved with a new refinement and maybe a second ligand could be added : -In Table S5 there are some missing classical statistics : I/σ(I), redundancy, number of reflections used for refinement, Bfactors and number of atoms for protein, ligands and water. Moreover, the number of unique reflections seems low (5,662) while in the PDB Report the number of reflections in test set is 1,994 (3.27% of total reflections). Please correct this point. Given statistics and poor quality of some parts of the structure (according to the PDB validation report) let me think that the resolution is overestimated. But without all statistic values (in particular I/σ(I)) it is difficult to decide (total and high resolution shell). -A clear panel with an omit map for the ligand is necessary but missing in Fig. 4. It should be added (ligand and density only, not surrounded residues) to clearly understand the conformation of the compound.
-As the structure is already available in PDB, I checked the density in the ligand binding pockets. The ligand in chain A is not fitting the density very well and the difference map indicates some issues. Moreover, the Bfactors are very high and the occupancy of the molecule should be refined. By this way, and by cutting the resolution, the authors would also be able to place the second molecule in chain B. Moreover, the negative difference density (in red) indicates that the acetyl moiety of nomilin is not at the right position, leading to the assumption that the compound in the ligand binding pocket could be the metabolite deacetylnomilin. Could it be possible ? -Some other issues : There are too many Ramachandran outliers. Side chain of Leu308 in chain B could be added. Lys453 in chain A is not correctly orientated and the big density blob between the two molecules in this region should be modelled with Glu461 B. Some water molecules could be added. Met323 A and B are not correctly orientated (maybe alternate positions). Cys284 B has an alternate position. A glycerol molecule could be added near Glu282. His407 seems to have alternate positions. Etc… -In sentence « From Fig. 4g, the biphenyl moiety in rifampicin interacts with M243 more closely than nomilin does, making rifampicin more sensitive to the local spatial variation introduced by M425Q mutation », if I understand well, it should be the same Met at the beginning and at the end, no ? -« These data suggest the nomilin activation on PXR may be different from other known PXR agonist. » Why is this ? What are the arguments ? Have the authors compared the structure of their complex with all other complex structures of PXR available in PDB ?
Reviewer #3 (Remarks to the Author): This manuscript by Fan et al. investigates an interesting question -whether a citrus fruit-derived small molecule, nomilin extends healthspan and lifespan in model organisms and the mechanism by which it occurs. The authors use a variety of different assays for this with some exciting results. However, there are some major concerns (listed below). 1. Figure 1: it looks like survival for 50 µM and 100 µM NML are comparable. Did the authors test higher doses than these? This would help understand NML-related toxicity (if any). 2. Figure 2: the death rates for each toxicant is very different depending on the mutant. What does this mean? Does NML require either nhr-8 or daf-12 for different toxicants? Where does this difference come from? Is it from the mechanism of action of the toxicant? 3. Figure 3: gst-10 RNAi data shows that NML requires gst-10 for lifespan extension. Yet, in Figure 2A, gst10 transcript levels are not significantly elevated by NML. Please explain why this is the case. 4. Figure 4: please identify on the figure that some of these data are related to human cells and some to worms. Presenting everything together can be very confusing. If not, they should be separated/ reorganized. 5. Figure 5: How does the D-galactose induced mouse model relate to the major themes of the manuscript? In other words, were detoxication or longevity genes/ proteins measured in this model in the brains? 6. Figure 6: BDL is not a liver toxicity model, more of a liver inflammation model. Why was not something like APAP or CCl4 not used if the goal was to investigate detoxication? Many of the mechanisms shown in 6H were not directly shown (eg. lifespan extension with NML in mice) therefore this diagram needs to be adjusted to showcase only what was proven with evidence during this study. 7. While the authors are correct in stating that "the increase in detoxication gene expression is a common transcriptomic signature in long-lived worms, flies and rodents", there is also strong evidence that many of these genes and proteins have higher expression and activity in diseased cells and organs. An example is NQO1 levels in Alzheimer's Disease brains. Therefore, one has to consider the context and dose under which upregulation of detoxication genes and proteins are beneficial. 8. The doses of nomilin that are needed to "attenuate the efficiency of therapy" in humans may be much larger than what was tested here. Therefore, it is very difficult to make this case.
Reviewer #4 (Remarks to the Author): Title: Pregnane X receptor agonist nomilin extends lifespan and healthspan through the detoxification functions Comments to author: This paper firstly used nematode C. elegans to demonstrate nomilin, a naturally occurring compound in citrus fruits, significantly extended the health span and toxin resistance. Further analysis indicated that the anti-senescence effect of nomilin is dependent on DAF-2/DAF-16 and NHR-30 8/DAF-12. Given hPXR was identified as the mammalian counterpart of NHR-8/DAF-12, the authors further identified that nomilin directly targets hPXR using X-ray crystallography and mutation assays. Finally, the authors demonstrated that nomilin exerts anti-aging effects on D-galactose-induced senescence mice and BDL mice. Overall, this study is interesting and well-organized. However, there are still some concerns that should be clearly addressed. Figure 4, the authors used the HEK293T cell-based reporter assay to demonstrate that hPPARα, hPPARβ, hPPARγ, FXR, LXRα and NRF2 can't be activated by nomilin. However, other nuclear receptors especially CAR shares high sequence homology with PXR. Whether CAR can be activated by nomilin should be further studied.

In the
2. RNA seq and QPCR analysis revealed that nomilin up-regulated the mRNA levels of hPXR target genes. What the effects of nomilin on the protein expressions of the PXR targets such as CYP3A11, UGT1A1 and SULT2A1?
3. The authors demonstrated that nomilin relieved the liver damage in BDL mice model. A positive control drug should be used to systematically assess the effect of nomilin in BDL-induced liver damage, such as mouse PXR agonist PCN. What's more, the effect of nomilin on liver damage is not sufficient to reflect its role in extending lifespan, other animal models should be used to prove that nomilin extends lifespan and healthspan, for example, senescence related mouse model. 4. There is a mistake in the figure legend of Figure 4, please check and revise carefully. Figure 6A, the scale bar is missing and not indicated in the figure legend. There is a mistake in the Figure 6H. The author should check and revise the diagram.

RESPONSE TO REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): In this paper, Fan et al investigate the properties of the citrus derived compound nomilin (NML) in nematodes and mice for a protective function in health and ageing.
NML robustly extends lifespan and appears to do so thought IIS pathway. They then focus on detoxification mechanisms and show that the beneficial effects of NML and nuclear DAF-16 localization depend on nhr-8 and daf-12. In fact, some detoxification genes were required for the NML longevity. Elegantly switching to structural analysis, the authors show that NML binds hPXR and use amino acid substitutions to assess NML interaction in its hPXR binding pocket. Next, they ask if hPXR and NML can affect worm survival. Further analyzing NML in mice, the authors show a neuroprotective effect using two paradigms.
This work shows an impressive breadth of methods and reports very interesting data.
Some of the conclusions, however, are not fully supported by the data and will require some additional experimentation. We thank the reviewer for the suggestive comments. We examined the mRNA expression of DAF-16 target genes. The results showed that the expression of DAF-16 target genes sod-2, sod-3, clk-2, acs-19 and lin-2 in C. elegans were increased by nomilin treatment, which support that the effects of nomilin is via IIS signaling. The data were added into Fig. 1f .
We have also performed additional experiment to see whether nomilin may induce dauer formation. We found that nomilin did not enhance the dauer formation either in the WT or in the daf-2(e1370) mutant background (Supplementary Figure S1q, r). We thought that the reason normilin mainly affected longevity instead of dauer formation, possibly because it targeted the intestinal cells and affected the local IIS activity ( Fig.1c, d). It was consistent with the report that the intestinal IIS pathway mainly regulates longevity, but not the dauer formation process, while the neuronal IIS  Table S6.
We thank the reviewer's suggestion. Following the reviewer's question, we made On the other hand, as the reviewer said, the overexpression of human PXR could not fully restore the lifespan extension effect of nomilin, possibly due to mammalian PXR could not fully activate the C. elegans target genes. Now we added these analyses and explanations into the text. 5. What is the effect of hPXR expression on the target genes of NHR-8 and/or DAF-12? This would be important to look at to support the notion that hPXR can substitute for the NHRs.
We performed the experiments to investigate whether hPXR could activate the target genes of NHR-8 and DAF-12. Nomilin-treated hPXR transgenic nhr-8 or daf-12 mutants were used to test the mRNA levels. The results showed that nomilin increased the expression of a few genes in these mutants (Fig. 5d, e), suggesting that hPXR partially compensates the function of worm NHR-8 and DAF-12. 6. In Fig 4H, the various hPXR mutants are analyzed are used for NML and RIF treatments. It would be great if the authors could comment on the fact that all mutants have a lower baseline activity. Would this suggest that the compounds stabilize the protein?
We are grateful to the suggestion from the reviewer. These data were obtained in same conditions. Thus, the possible explain for the lower baseline activity of hPXR mutants is that the activities of the mutants in response to the endogenous agonists may be lower than those of wild type hPXR. Currently, we are no evidence to show that the compounds stabilize the protein. Now we added a short comment to the text. We fully agree to the comments. We have shown that the effects of nomilin on BDL damage were attenuated in the liver of PXR knockout mice (Fig. 9a). Now we carried out D-galactose induced senesence in PXR knockout mice, and treated with nomilin.
The results showed that nomilin did not improve the cell death in the brain, motor deficts as well as inflammatory infiltration in the liver of PXR knockout mice induced by D-galactose, supporting that NML activates the detoxification pathway in the context of neuroprotection. Now the data were added as Fig. 8.

Minor points
1. While the language if clear, I would suggest for a native speaker to edit the manuscript.
We asked a native English speaker to revise the language.
2. I would recommend refraining from using the term "anti-aging". This is not very well defined, particularly in the context of the paper where survival is only measured in the worm. The term better belongs into popular culture.
We changed "anti-aging" to "aging inhibiting" or "longevity intervention" through the text.
3. Also from the data presented here, I think one cannot conclude any recommendations or comments regarding citrus consumption.
We revised the relevant conclusion.
Reviewer #2 (Remarks to the Author): In manuscript "Pregnane X receptor agonist nomilin extends lifespan and healthspan through the detoxification functions", the authors show through a combination of Xray structure, in vitro and in vivo (in nematode and mouse) experiments that nomilin, a compound found in citrus fruits, is able to activate in a beneficial way for the organism the nuclear receptors NHR8 and DAF12 in worms, as well as their couterpart in mammals, PXR. PXR is a receptor rather known for its adverse effects following the binding of exogenous molecules, whereas here the authors show that nomilin can increase lifespan and healthspan via PXR. From a technical point of view, the biological experiments are numerous and varied, and are rigorously carried out with all necessary controls. The accomplished work seems dense and of quality, even if the authors sometimes get confused in the text, especially for the notations of the mutants. However the quality of the X-ray structure is a major issue. It can be improved and needs to be re-refined. My comments will essentially focus on the structural aspects of this manuscript, even though I have some general comments, listed below, to improve the readability : 1/ Please specify the origin of PXR in the whole manuscript, by writing mPXR (for mouse) or hPXR (for human) depending on the sentence or figure legend.
We thank the reviewer for the suggestion. Now we specified the origin of PXR through the text. Fig. 4 : panels D, E, F, and G are not easy to read, please increase the size of residue labels, and potentially the size of these panels. In G, write the name of the compounds with the same color code as the sticks and place the labels in a more obvious way. In H, there is a mistake, M245Q should be M425Q, and M247A should be S247R. In C, please label the b1' strand as it is cited in the text.

2/
Molecules in panel A are too small to be read correctly.
We have re-made the graph and corresponding labels and corrected the mistakes. We corrected the mistakes. 7/ In « Mammalian PXR is a functional ortholog of NHR-8 /DAF-12 » section there is a mistake on mutant that should be hPXR S247R (I expect) We corrected the mistakes. 8/ Maybe I missed something, but in « Dissussion », what are AD, PD and CR ?
We revised "AD, PD and CR" as "Alzheimer's disease, Parkinson's disease and caloric restriction".
Here are my scientific comments : 9/ For TR-FRET assay, why the authors used the T0901317 LXR agonist as a reference instead of rifampicine or SR12813 that are commonly used as PXR reference ligands ? Is this compound known to be a specific PXR agonist ?
T0901317 is also a high-affinity ligand for PXR, which was suggested by the However, several points let me think that this structure should be improved with a new refinement and maybe a second ligand could be added : -In Table S5  Please correct this point.
Given statistics and poor quality of some parts of the structure (according to the PDB validation report) let me think that the resolution is overestimated. But without all statistic values (in particular I/σ(I)) it is difficult to decide (total and high resolution shell).
We thank the reviewer for the suggestion and re-processed the diffraction data for a better data reduction and refinement. We discarded several image with poor diffraction and re-scaled the data to a resolution up to 2.1 Å, which is slightly lower than the one in the original manuscript but have the statistics improved significantly. We have renew the crystallographic table with all the statistics required. In addition, we have attached the coordinate file and the reflection file in this revision for further evaluation.
-A clear panel with an omit map for the ligand is necessary but missing in Fig. 4. It should be added (ligand and density only, not surrounded residues) to clearly understand the conformation of the compound.
We have made the omit map for the ligand as figure 4h according to the suggestion.
-As the structure is already available in PDB, I checked the density in the ligand binding pockets. The ligand in chain A is not fitting the density very well and the difference map indicates some issues. Moreover, the Bfactors are very high and the occupancy of the molecule should be refined. By this way, and by cutting the resolution, the authors would also be able to place the second molecule in chain B.
Just as the reply to previous question, we have re-processed the data with a slightly lower resolution for a improved refinement. In the revised model, we successfully fit the ligands in both protomers by occupancy refinement, with a better electron density map in protomer A than that in B (Fig. 4h). We appreciate the reviewer's suggestion and it help us make more accurate model.
Moreover, the negative difference density (in red) indicates that the acetyl moiety of nomilin is not at the right position, leading to the assumption that the compound in the ligand binding pocket could be the metabolite deacetylnomilin. Could it be possible ?
We have confirmed the compound structure by a mass spectrometry assay, which showed that the compound is exact nomilin, but not deacetylnomilin (see below Figure   1&2 and Table 1).   -Some other issues : There are too many Ramachandran outliers.
Side chain of Leu308 in chain B could be added.
Lys453 in chain A is not correctly orientated and the big density blob between the two molecules in this region should be modelled with Glu461 B.
We agreed and have made the modifications accordingly.
Some water molecules could be added.
Met323 A and B are not correctly orientated (maybe alternate positions).
Cys284 B has an alternate position.
A glycerol molecule could be added near Glu282.
His407 seems to have alternate positions.

Etc…
We We have made a superposition among the hPXR bound with nomilin, rifampicin, SR12813, Hyperforin, and clotrimazole, as well as its empty state ( Supplementary   Fig. S4a, b ). In the superposition between hPXR bound with nomilin and rifampicin, we found the helix of aa 193-209 has a spatial clash with rifampicin. Although this helix can only be visible in our structure, the superposition showed that when bound with rifampicin, there has to be a displacement in this region to avoid clash with the ligand, implying a significant structural difference between hPXR bound with nomilin and rifampicin.  3. Figure 3: gst-10 RNAi data shows that NML requires gst-10 for lifespan extension.
Yet, in Figure 2A, gst10 transcript levels are not significantly elevated by NML.
Please explain why this is the case.
Gst-10 is a typo. We are sorry for the mistake. We selected to knockdown the nomilin upregulated gene Gst-4 (Fig. 2a) to verify the pathway. Now we corrected the mistake.
4. Figure 4: please identify on the figure that some of these data are related to human cells and some to worms. Presenting everything together can be very confusing. If not, they should be separated/ re-organized.
We re-organized the Figure as We thank the reviewer for the helpful suggestions. The liver is a major detoxification organ in mammals, which may detoxify D-galactose, and lower the concentration of D-galactose in circulation and the organs. However, PXR is also expressed in the brain, which may play detoxification functions. We analyzed PXR target gene expression and showed that nomilin increased the expression of Gsta1, Gsta2, Mdr3, Cyp8b1, Cyp27a1 and Cyp2d22 in the hippocampus, suggesting that nomilin may also increase PXR signaling in the brain of mice. Now the data were added as Supplementary Fig. S6d. 6. Figure 6: BDL is not a liver toxicity model, more of a liver inflammation model. Cholestatic PXR knockout mice exhibits more hepatic damage than wild-type mice both after bile duct ligation and cholic acid feeding [4][5][6]. PXR agonist reduces lithocholic acid-induced liver injury in wild-type mice, but not in PXR knockout mice, via the upregulation of UGT1A1, MRP2, MRP3 and CYP3A4 facilitating bilirubin elimination and detoxification of bile acids [6,7].
The summary diagram has been revised accordingly.
7. While the authors are correct in stating that "the increase in detoxication gene expression is a common transcriptomic signature in long-lived worms, flies and rodents", there is also strong evidence that many of these genes and proteins have higher expression and activity in diseased cells and organs. An example is NQO1 levels in Alzheimer's Disease brains. Therefore, one has to consider the context and dose under which upregulation of detoxication genes and proteins are beneficial.
We really appreciate the reviewer's suggestion. As the reviewer said, in many biological processes, the specific context of application and related gene/protein dosage is critical for their beneficial function. For example, the elevation of NQO1 is associated with AD pathology, but it is commonly viewed as a neuroprotective response to the oxidative stress that accompanies AD (1,2). Therefore, a causative analysis is needed to identify the positive/negative role of an elevation of gene/protein in a specific biological process. That's why in this manuscript, we have shown that NHR-8/DAF-12/PXR activation was not only related, but also required for the lifespan extension and detoxification process in C. elegans and mice. 8. The doses of nomilin that are needed to "attenuate the efficiency of therapy" in humans may be much larger than what was tested here. Therefore, it is very difficult to make this case.
We thank the reviewer for pointing this out. Although the citrus juices and grapefruit juices contain high concentrations of limonoids (320 ppm and 195 ppm), the effects of nomilin on drug metabolism remains unclear. Now we are working on this, hopefully would answer the question. We revised the sentence to "Whether the consumption of nomilin-containing citrus fruits and juices change drug metabolism needs to be investigated".
Reviewer #4 (Remarks to the Author): Title: Pregnane X receptor agonist nomilin extends lifespan and healthspan through the detoxification functions Comments to author: This paper firstly used nematode C. elegans to demonstrate nomilin, a naturally occurring compound in citrus fruits, significantly extended the health span and toxin resistance. Further analysis indicated that the anti-senescence effect of nomilin is dependent on DAF-2/DAF-16 and NHR-30 8/DAF-12. Given hPXR was identified as the mammalian counterpart of NHR-8/DAF-12, the authors further identified that nomilin directly targets hPXR using X-ray crystallography and mutation assays.
Finally, the authors demonstrated that nomilin exerts anti-aging effects on Dgalactose-induced senescence mice and BDL mice. Overall, this study is interesting and well-organized. However, there are still some concerns that should be clearly addressed.
1. In the Figure 4, the authors used the HEK293T cell-based reporter assay to demonstrate that hPPARα, hPPARβ, hPPARγ, FXR, LXRα and NRF2 can't be activated by nomilin. However, other nuclear receptors especially CAR shares high sequence homology with PXR. Whether CAR can be activated by nomilin should be further studied.
We thank the reviewer for pointing this out. We created pcDNA3.1-hCAR expression plasmid and performed reporter gene assay. The results showed that nomilin did not activate hCAR transactivity. The data was added as Supplememntary Fig. S3b. 2. RNA seq and QPCR analysis revealed that nomilin up-regulated the mRNA levels of hPXR target genes. What the effects of nomilin on the protein expressions of the PXR targets such as CYP3A11, UGT1A1 and SULT2A1?
We agree with the comments from the reviewer. We carried out western blots of PXR targets Gsta1, Cyp3a11 and Cyp51a1 in the livers in D-gal-treated mice, and the results showed that nomilin also increased the protein expression, which consistent to the RT-PCR results. The data were added as Supplememntary Fig. S5b, c. 3. The authors demonstrated that nomilin relieved the liver damage in BDL mice model. A positive control drug should be used to systematically assess the effect of nomilin in BDL-induced liver damage, such as mouse PXR agonist PCN. What's more, the effect of nomilin on liver damage is not sufficient to reflect its role in extending lifespan, other animal models should be used to prove that nomilin extends lifespan and healthspan, for example, senescence related mouse model. The chemotherapeutic drug doxorubicin may induce accelerated aging in patients, and has been used to induce senescent animal models in aging research. Thus, we used doxorubicin-induced accelerating aging mouse model to test the effects of nomilin on lifespan and healthspan. The results showed that nomilin could improve the lifespan and movement ability, liver damage and heart fibrosis in doxorubicin-treated mice, which further support that nomilin exerts aging-retarding effects via the detoxification function. The data were shown as Fig. 7. 4. There is a mistake in the figure legend of Figure 4, please check and revise carefully.
We revised the mistakes. Figure 6A, the scale bar is missing and not indicated in the figure legend.

In the
There is a mistake in the Figure 6H. The author should check and revise the diagram.
We added the scale bar to the Fig. now as Fig. 9a, and the diagram was revised.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): All points have been addressed and the paper was enhanced by significant new data. I am in full support of publication.
Reviewer #2 (Remarks to the Author): I thank the authors for implementing the majority of the corrections I suggested and for their effort to re-process the structural data. The quality of the structure has been improved. However I noticed remaining major concerns.
1/ The alternate position of His407, which is clearly indicated by the difference electron density, is still missing. I insist on that point because of the location of this residue, near the ligand, which is known to adopt several orientations depending of the ligand bound to PXR. Moreover the authors modeled one orientation in one chain, and the other one in the other chain of the asymmetric unit.
2/ The authors added an argument supported by Supp Fig. S4a to discuss the difference of activation levels observed for nomilin and rifampicine respectively (line 246 p7). "In addition, one of the major structural differences between hPXR bound with nomilin and rifampicin is the helix formed by amino acids 193-209, which is well-refined in our structure but absent in a previous report (Supplementary Overall, the authors have made a significant effort to address the reviewer's concern and thereby substantially improved the manuscript. However, some points still need clarification as they have not been corroborated sufficiently. Major points: 1. One major concern is that the authors should explain why they choose doxorubicin-induced accelerating aging mice as the senescence model. In the main text, the authors proposed "This drug has been used to induce senescence in an animal model in aging research [72]". However, the cited reference is "An evaluation of hepatotoxicity in breast cancer patients receiving injection Doxorubicin", which is about the hepatotoxicity and doxorubicin. Why did the authors not use more classical senescence mouse models, such as SAMP or naturally aging mice. Additionally, in the Figure 7, nomilin promoted the survival of doxorubicin-treated mice and suppressed inflammatory cell infiltration in the liver. However, these data only suggested that nomilin ameliorated the liver injury induced by doxorubicin. At least, the author should perform the analysis of senescence-associated secretory phenotype, for example, p16ink4a, IL1α, and IL6. 2. In Fig.6, the authors used D-gal to induce senescence. However, there is still a lack of behavioral experiments to reflect the hippocampus and neuromuscular functions.

Response to the reviewer's comments
Reviewer #1 (Remarks to the Author): All points have been addressed and the paper was enhanced by significant new data. I am in full support of publication.
We thank the reviewer for the positive comments.
Reviewer #2 (Remarks to the Author): I thank the authors for implementing the majority of the corrections I suggested and for their effort to re-process the structural data. The quality of the structure has been improved. However I noticed remaining major concerns.
1/ The alternate position of His407, which is clearly indicated by the difference electron density, is still missing. I insist on that point because of the location of this residue, near the ligand, which is known to adopt several orientations depending of the ligand bound to PXR. Moreover the authors modeled one orientation in one chain, and the other one in the other chain of the asymmetric unit.
We thank the reviewer for the suggestion, and we have re-processed the data to add the alternative conformation of H407, in both protomers. We have modified Figure 4D with the alternative conformations, and the RCSB deposition has been updated accordingly.
2/ The authors added an argument supported by Supp Fig. S4a to discuss the difference of activation levels observed for nomilin and rifampicine respectively (line 246 p7). "In addition, one of the major structural differences between hPXR bound with nomilin and rifampicin is the helix formed by amino acids 193-209, which is well-refined in our structure but absent in a previous report ( Supplementary Fig. S4a)  We thank the reviewer for pointing out the incorrect description in the main text and the mistake in Figure S4a. We have revised the paragraph to accurately reflect the correct information and have updated Figure S4a accordingly. We apologize for any confusion caused by the previous incorrect description. We are grateful for the reviewer's input, which has helped improve the accuracy and integrity of our study.
I also noticed some other minor points.  (1). While in a 2004 G&D paper, DAF-12 was also reported to have an similar 3' halfsite A-G-T-T/G-C-A/G DNA binding sequence (2). Though we did not find any report about the binding site of NHR-8, we did find a recent Cell Metabolism paper reporting that " NHR-8 and DAF-12 share significant homology in DNA-and ligand-binding domains (DBD; LBD), and have identical residues in the P-box, a motif in the first zinc finger that functions in DNA recognition " (3). Therefore, we think these may explain why hPXR could patially rescue the daf-12(-) mutant ( Figure 5). We thank the reviewer for the helpful suggestions. Now we added it to the manuscript on Page 8. 5/ Just a remark, the mass spectrometry control should have been done directly on PXR/nomilin crystals as the compound could have been modified during the crystallization process. It happens some times.
"We have confirmed the compound structure by a mass spectrometry assay, which showed that the compound is exact nomilin, but not deacetylnomilin (see below Figure 1&2 and Table 1)." We appreciate the reviewer's comment regarding the potential chemical modification of nomilin during the crystallization process. As per the recommendation, we have remade the crystallization trial and conducted mass spectrometry analysis directly to ensure the integrity of the compound during the crystallization process. Our results confirm that there was no significant deacetylation detected, validating the findings reported in our previous revision.
The data were shown as Supplementary Methods- Figure 1a-d and Table 1. Thank you for bringing this to our attention and allowing us to further verify the robustness of our experimental approach.
We thank the reviewer for pointing out this typo, and it has been corrected in the revised manuscript.
We have changed W299 to W299R in Fig. 4i.
Reviewer #3 (Remarks to the Author): Here are my comments to the author responses -1. Please increase the quality of the supplemental figures so that the details of figures can be clearly seen.
We have prepared high resolution figures. We are grateful to the suggestions. Now we have switch Fig. 7 and 8. We thank the reviewer for the comments and suggestions. Now we have added the Kaplan-Meier survival curve and corresponding statistical analysis (Long-rank test) as Supplementary Fig. S7.
We labeled the trichrome figures and provided figures that show comparable regions for all groups (Fig. 8h).
We provided additional gait parameters in PXR -/mice ( Fig. 7e-f). However, after two-weeks treatment, doxorubicin mice showed significant body weight reduction and poor physical condition that prevented them from completing subsequent behavioral experiment, so we could not obtain the gait parameters in this model.
We provided body weight data for all four mouse models. We also provided the food consumption data of three models ( Supplementary Fig. S9). However, for doxorubicin-treated mice, the food was soaked in water to facilitate their eating since the mice were very weak. Thus, the food intake cannot be count accurately in this model.

OK
Reviewer #4 (Remarks to the Author): Overall, the authors have made a significant effort to address the reviewer's concern and thereby substantially improved the manuscript. However, some points still need clarification as they have not been corroborated sufficiently.
Major points: 1. One major concern is that the authors should explain why they choose doxorubicin-induced accelerating aging mice as the senescence model. In the main text, the authors proposed "This drug has been used to induce senescence in an animal model in aging research [72]". However, the cited reference is "An evaluation of hepatotoxicity in breast cancer patients receiving injection Doxorubicin", which is about the hepatotoxicity and doxorubicin. Why did the authors not use more classical senescence mouse models, such as SAMP or naturally aging mice.
Additionally, in the Figure 7, nomilin promoted the survival of doxorubicin-treated mice and suppressed inflammatory cell infiltration in the liver. However, these data only suggested that nomilin ameliorated the liver injury induced by doxorubicin. At least, the author should perform the analysis of senescence-associated secretory phenotype, for example, p16 ink4a , IL1α, and IL6.
We thank the reviewer for the comments and suggestions. Doxorubicin can induce cellular and organ senescence in many aspects as well as the biomarkers of senescence such as p16 INK4A , p21 and β-galactosidase in rodents and humans (1,2). This model was wildly used in aging research. We are sorry for inappropriate reference citing. Now we have cited relevant references in the text.